We propose to continue operation of the Center for Eukaryotic Structural Genomics (CESG) as a Specialized Center under Phase-2 of the Protein Structure Initiative (PSI). Our focus will remain on eukaryotic targets. About 60% of its structures will be directed toward enlarging regions of fold-space covered primarily by proteins from humans and other higher organisms;20% will be aimed at proteins of biomedical relevance;and 20% will be in response to requests from the community. Specialized Center funding will necessitate downsizing through reductions in technical support positions and junior Ph.D. positions. CESG will retain staff and students capable of continuing its research and development projects aimed at increasing success rates and lowering costs of steps involved in producing eukaryotic proteins and solving their structures by X-ray crystallography and NMR spectroscopy. We will carry out research and then test promising new approaches in the context of a high-throughput pipeline. We expect to produce 50 structures per year by the third year of PSI-2. We see opportunities for improving technology and lowering costs at multiple stages. Major areas for research and development will include (1) vector designs to lower costs and permit transfer of target genes between expression plasmids used by CESG's cell-based (E. coli) and cell-free (wheat germ extract) protein production pipelines, (2) improved small-scale screening for protein production, solubility, and stability, (3) more efficient crystallization and crystal optimization, (4) identification of optimal conditions for NMR structure determinations, and (5) automation of data collection, structure determination, refinement, and validation. CESG captures detailed information about all of its activities, which it uses in evaluating new technology. CESG will conform to the PSI rules for releasing structures and for making the products of the project freely available. CESG will hold workshops and training programs. Activities of the Center will be monitored and guided by an advisory committee representing both biological and technical expertise.